Effect of nitrogen fertilizer on seed yield and quality of Kengyilia melanthera (Triticeae, Poaceae)

Widely distributed in the alpine sandy grassland in east Qinghai-Tibet Plateau (QTP), Kengyilia melanthera is considered as an ideal pioneer grass for the restoration of degraded and desertification grassland in the region. Under the special ecological and climatic conditions in the northwest Sichuan plateau located in east QTP, it is of great significance to optimize the amount of nitrogen fertilizer for the seed production of this species. The impact of nitrogen (N) fertilizer application on seed yield and quality of K. melanthera ‘Aba’, the only domesticated variety in the Kengyilia genus of Poaceae, was investigated based on two-year field experiments in the northwestern Sichuan plateau. The results showed that with the increase of N fertilizer application, the number of tillers, number of fertile tillers, 1,000-seed weight and seed yield of this species increased likewise. The optimum N fertilizer rate deduced in the present study was 180 kg·hm−2, where the number of fertile tillers 1,000-seed weight and seed yield reached the peak values. Interestingly, the standard germination rate, germination energy, accelerated aging germination rate, dehydrogenase and acid phosphatase activity of seeds were not affected by the increasing the input of N fertilizer. The comprehensive evaluation of membership function showed that the optimal N fertilizer treatment was 180 kg·hm−2 both for 2016 and 2017. This study provided a certain practical suggestion for the improvement of seed production of K. melanthera in the northwest Sichuan plateau.


INTRODUCTION
The Qinghai-Tibet Plateau (QTP), the so-called third pole of the world, is an important eco-region. The grassland ecosystem in the QTP is undergoing extensive and longtime deterioration and even desertification, due to both human and natural factors (Lu et al., 2017;Wu, Yu & Jin, 2017). Since the beginning of the 21st century, the Chinese government has implemented large-scale ecological restoration projects to conserve

Experimental sites
The experiment was conducted for two consecutive years, between 2015 and 2017. The field is located in Hongyuan county, Aba Autonomous Prefecture, Sichuan province, a semi-humid region of the eastern QTP of China (31 79′35″N, 102 55′47″E, 3,480 m a.s.l.). Before sowing, the grasses and weeds were eliminated in all plots by hand weeding and herbicide spray, then the field was ploughed twice. The soil of experimental land was a subalpine meadow soil in nature, and chemical properties were provided in Table S1. In the 0-30 cm soil layer (pH 6.02), the concentration of organic matter, total nitrogen, total phosphorus, total potassium, available nitrogen, phosphorus, and potassium in the soil was 13.8, 1.32, 0.86, 0.68, 87.47, 62.55, and 227.46 mg·kg −1 , respectively.

Experimental management
Seeds of K. melanthera (variety named 'Aba') was supplied by the Sichuan Academy of Grassland Sciences, and was harvested in the most recent production year before the field plot experiment. Its 1,000-seed weight and germination rate were 4.97 g and 80%, respectively. Generally, during the sowing year of (2015), the vast majority of K. melanthera plants growth, development, flowering, and seed setting are markedly reduced by the continental plateau's cold temperate monsoon climate. Hence, the seed yield and quality data in this study were recorded beginning from the second year (2016).

Experimental design
The fertilizer treatments (urea fertilizer, 46% N) were applied to the experimental plots during the elongation stage, in mid June of 2016 and 2017. This trial is adopted as was a randomized complete block design for eight sample plots with different amounts of nitrogen fertilization (N 1 -0 kg·hm −2 , N 2 -60 kg·hm −2 , N 3 -90 kg·hm −2 , N 4 -120 kg·hm −2 , N 5 -150 kg·hm −2 , N 6 -180 kg·hm −2 , N 7 -210 kg·hm −2 , N 8 -240 kg·hm −2 ). Each N fertilizer treatment had four replications' plots and a total of 32 plots. Sowing was performed by the drill method on May 20, 2015, with a seed density of 22.5 kg·hm −2 , sowing depth of 1-2 cm, and 50-cm row spacing. Experimental units consisted of plots with an area of 20 m 2 (5 m × 4 m) and 8 rows, and seeds were not sown within 50 cm of the boundary of the plot in order to reduce the marginal effect.

Seed yield components
The number of tillers (NTs), number of fertile tillers (NFTs), number of spikes (NSPs), number of florets (NFLs), number of fertile florets (NFFLs), and 1,000-seed weight (TSW) were measured to evaluate the seed yield components. At full-blooming stage (the proportion of plants that flowered reached 50%), a row was randomly selected from each sample plot for measurement of the NTs and NFTs, and four replicate measurement rows were performed within each plot. Here the length of selected row is 1 m and not on border rows of plot. In addition, the different indices cannot be sampled from the same plant. The NSPs, NFLs, and NFFLs were counted based on ten randomly sampled tillers in full-blooming stage from each plot. The seeds from each plot were harvested from the 30th day after peak anthesis. 1,000 sun-dried seeds were randomly selected to determine the TSW, four replications were carried out.

Seed yield
The seed yield indices could be represented by the harvested seed yield (HSY), potential seed yield (PSY), and presentation seed yield (PRSY). At the 30th day after peak anthesis, for each measurement, four row-segments with the length of 1 m were randomly selected from each plot, and all its seeds were harvested. After these seeds were threshed, cleaned, and naturally sun-dried to a seed moisture content of about 150 g·kg −1 , and the HSY of each N treatment could be obtained. The PSY and PRSY per unit area were calculated with the following formulas:

Seed quality
To reduce or avoid dormancy and dormancy and after-ripening of freshly harvested seeds, seed quality trait analysis was tested approximately two months following harvest. The seed quality indices were based on standard germination rate (SGR), germination energy (GE), accelerated aging germination rate (AAGR), dehydrogenase (DE) and acid phosphoesterase (APH) activity. Because some weed seeds might be mistakenly harvested when K. melanthera seeds were harvested, so these seeds should be removed, the remaining seeds are called pure seeds. We randomly selected 50 pure seeds under different N treatments respectively and placed them into a Petri dish with two layers of filter paper on the bottom, adding a suitable amount of distilled water (preferably submerging seeds), and then moved them to cultivate in a plant growth chamber at a day temperature of 25 C (16 h) and night temperature of 16 C (8 h). Finally, we determined the SGR and GE of seeds on the 4th and 7th days of seed germination, respectively. For the seed-aging treatment, 250 pure seeds were randomly selected and embedded into an aging tank kept at 41 C (72 h). The AAGR was measured after aging treatment, measurements from SGR and this method were identical. The DE and APH activity of seeds were determined by the triphenyl tetrazolium chloride (TTC) (Mao et al., 2001) and the sodium p-nitrophenol phosphate method (Stephen, Daniel & William, 1989), respectively. The assays described above were repeated four times.

Statistics analysis
The statistical analysis was conducted by one-way and two-way analysis of variance (ANOVA) using IBM SPSS Statistics 20.0 software. The two-way analysis of ANOVA with a general linear model at P < 0.05 was applied to determine the combined influence of N fertilizer and trial years on seed yield components, seed yield, and quality. Bonferroni adjustment was applied after multiple hypothesis testing at the P < 0.05 probability level.
TBtools software is used to draw heat maps and other graphs were visualized using GraphPad Prism 8 procedure.
The membership function method was used to explore the response of seed yield components, yield, and quality to N fertilizer, and to determine the best fertilizer application rate for K. melanthera. The calculation formula for the forward membership function was y ¼ x a À x min ð Þ = x max À x min ð Þ , and the negative membership function was y ¼ 1 À x a À x min ð Þ = x max À x min ð Þ , where the x a is the value of certain index, and the x max and x min represent the maximum and minimum values in the same index of seed traits.

RESULTS
The influence of N fertilizer on seed yield components Because of a typical plateau continental climate, plant growth and flowering of K. melanthera were restrained in the sowing year (2015). Therefore, we collected the seed yield-related and quality-related data from 2016 and 2017. N fertilizer had a significant influence (P < 0.05) on some yield components of seed including number of tillers (NTs), number of fertile tillers (NFTs), number of fertile florets (NFFLs) per spike, and 1,000-seed weight (TSW, Table 1). And non-significant difference was observed in all indices detected in 2016 and 2017.
Compared to 0 kg·hm −2 (N 1 , check), the NTs and NFTs in both trial years were significantly increased when 90-240 kg·hm −2 (N 3 -N 8 ) of N fertilizer was applied (Fig. 1A).  It is worth noting that we observed a marginal decline (P > 0.05) in NTs, NFTs, NFFLs per spike, and TSW when the application amount of N fertilizer was over N 7 in 2016 and 2017, which may be due to the negative effect of excess N fertilizer.

The influence of N fertilizer on seed yield and quality
The doses of N fertilizer application had a significant influence on the seed yield (Table 2, P < 0.05), but a non-significant effect in improving seed quality (standard germination rate (SGR), germination energy (GE), accelerated aging germination rate (AAGR), dehydrogenase (DE) and acid phosphoesterase (APH) activity, P > 0.05). With increasing rates of N fertilizer application rate, three seed yield indices (harvested seed yield (HSY), potential seed yield (PSY), and presentation seed yield (PRSY)) showed a tendency of first increasing and then decreasing (Figs. 2A and 2B), and reached their peak values under N 6 treatment in both 2016 and 2017. What is noteworthy is that excessive rates of N fertilizer application (N 7 -N 8 ) had a non-significant suppression or improvement on seed quality, however, was accompanied by a reduction in three seed yield indices (Fig. 2, Table 3). During two-year independent field trials, we found that seed yield in 2017 was higher than that in 2016 under the same N fertilizer treatment, but the difference was non-significant.
The relationship of N application and seed yield or seed components N application had a significant influence on seed yield of K. melanthera, which could be explained by the increase of seed quantity and weight under the N effect (Table 4). By means of path analysis, we found that seed yield was significantly correlated with number of fertile tillers/m 2 (NFTs/m 2 , r = 0.944, P < 0.05), 1,000-seed weight (TSW, r = 0.912, P < 0.05), and number of fertile florets (NFFLs) per spike (r = 0.899, P < 0.05), but not with number of spikes (NSPs) per fertile tiller (r = 0.806, P > 0.05) and number of florets (NFLs) per spike (r = 0.818, P > 0.05). Also, seed yield (r = 0.909, P < 0.05), NFTs/ m 2 (r = 0.950, P < 0.05) and TSW (r = 0.921, P < 0.05) were observably associated with N treatment. It was therefore evident that N application was one of the most effective ways to enhance seed yield of K. melanthera. For box plots, top and bottom of box represent 75th and 25th percentiles, respectively. Different lower-case letters above the column and curve indicate significant differences under different N fertilizer treatments in each trial year (Bonferroni, P < 0.05). Vertical bar represents the standard error of mean. Statistical significance was determined as Ã P < 0.05, ns was P > 0.05. Note: N 1 -N fertilizer 0 kg·hm −2 (Control); N 2 -N fertilizer 60 kg·hm −2 ; N 3 -N fertilizer 90 kg·hm −2 ; N 4 -N fertilizer 120 kg·hm −2 ; N 5 -N fertilizer 150 kg·hm −2 ; N 6 -N fertilizer 180 kg·hm −2 ; N 7 -N fertilizer 210 kg·hm −2 ; N 8 -N fertilizer 240 kg·hm −2 . Full-size  DOI: 10.7717/peerj.14101/ fig-1 Comprehensive analysis of membership function and heat map The changes in the seed trait indices of K. melanthera represented a certain degree of correlation and difference based on the N fertilizer treatments, and a single index cannot fully explain the influence of N fertilizer treatment on plant reproduction and seed production. Consequently, it was necessary to conduct a comprehensive evaluation of multiple indices to determine the optimal amount of N fertilizer application. Based on the values of each seed trait index measured under different N treatments, including indices of six seed yield components, three seed yield, and five seed quality, to calculated the membership function values (Table 5). Higher membership function values represented more distinct improvement of K. melanthera seed yield or quality. The results indicated that the optimal deses of N fertilizer treatment was N 6 (followed closely by N 7 ) in both 2016 and 2017 (Table 5 and Table S2). Up-regulation or down-regulation of the indices for seed yield components, seed yield, and seed quality were revealed by the heatmap and hierarchical cluster analysis. In the first trial year, up-regulation of all indices was most obvious for N 6 , followed by N 5 and N 7 (Fig. 3A). In addition, N 5 , N 6 , N 7 , and N 8 could be classified into one group through hierarchical cluster analysis. Meanwhile, we also noted that germination energy (GE) and acid phosphoesterase (APH) revealed significant up-regulations at N 3 and N 4 , respectively. In the second trial year, up-regulation of most indices were relatively significant for both N 6 and N 7 , and could be classified into one group through cluster analysis (Fig. 3B). Interestingly, accelerated aging germination rate (AAGR) and standard germination rate (SGR) exhibited down-regulation at N 6 . Therefore, the heat maps obtained the same results as the comprehensive analysis of membership function in both trial years, that is N 6 (180 kg·hm −2 urea) was the optimal N fertilizer treatment.

Effect of N on seed yield
The main features of elite varieties of perennial grass are known to increase the yield of aboveground biomass and vegetation coverage of grassland stands thereby meets the needs of grassland husbandry and ecological restoration (Varadi, Kadar & Duda, 2019). Numerous investigations of increasing seed yield through different agronomic management practices have been carried out for many perennial grasses in the QTP, e.g., Elymus sibiricus, E. nutans, and Phalaris arundinacea, but few have probed into the effects of fertilization on seed yield. It is generally believed that a low fertilization rate results in seed yield not reaching the desired goal, but excess fertilizers led to increasing environmental pollution and reducing of seed yield (Zhang, 2017). Sufficient nitrogen (N) in the soil is the basis for high seed yield. Seed yield is significantly positively correlated with N fertilizer dose within a specific range, even if no other fertilizer is applied (Wang et al., 2005a). Most researchers choose N fertilizer to increase seed yield as it is the most consumable component during plant growth and seed maturation (Li et al., 2018b).
In addition, N fertilizer can not only improve the developmental structure of plant roots and absorption of nutrients, but also the formation and size of grains, which can directly determine the seed yield, were promoted during later stages of plant growth . Therefore, compared with other fertilizers, N fertilizer is one of the most effective measures to improve seed yield (Oliveira et al., 2007). By combining seed yield indices (harvested seed yield (HSY), potential seed yield (PSY) and presentation seed yield (PRSY)) under different N application doses (N 1 -N 6 ), we propose an optimal rate of N fertilizer application (N 6 ) affected seed yield in K. melanthera (see in Figs. 2A and 2B). Similar results could be observed with an increase in the number of seeds or the weight of a Measurement results are means ± standard error (n = 4). In the same column, standard error of mean followed by the same lower-case letters indicate no-significant differences under different N fertilizer treatments in each trial year base on a Bonferroni multiple comparison test at P < 0.05. SGR, standard germination rate; GE, germination energy; AAGR, accelerated aging germination rate; DE, dehydrogenase; APH, acid phosphoesterase; N 1 -N fertilizer 0 kg·hm −2 (Control); N 2 -N fertilizer 60 kg·hm −2 ; N 3 -N fertilizer 90 kg·hm −2 ; N 4 -N fertilizer 120 kg·hm −2 ; N 5 -N fertilizer 150 kg·hm −2 ; N 6 -N fertilizer 180 kg·hm −2 ; N 7 -N fertilizer 210 kg·hm −2 ; N 8 -N fertilizer 240 kg·hm −2 . single seed (Martiniello, 1998). The reason may be that the N accumulated in the vegetative organs is transported and redistributed to the grains, and thus enhancing seed yield due to fuller grains (Yi et al., 2020). As N fertilization continues to rise, seed yield indices were significantly decreased from N 7 to N 8 . This may have been caused by excessive growth of competing plants encouraged by excessive N fertilizer application, or by dehydration of plant roots (Aly, 1993). It happens that there is a similar case, a study of a wildrye Elymus nutans in Tibet, which showed a peak yield of 2016 kg·hm −2 at a N fertilizer dose of 250 kg·hm −2 , and a trend of falling continuously for HSY when the N fertilizer dose was continued increased (Song et al., 2008). Therefore, we should manage proper N fertilizer rate to optimize N-uptake in seed production of K. melanthera.

Effect of N on seed yield components
For most grass species, seed yield always depends on the multiplicative effect of the yield components (number of tillers (NTs), number of fertile tillers (NFTs), number of spikes (NSPs), number of florets (NFLs), number of fertile florets (NFFLs), and 1,000-seed weight (TSW)) (Dewitt et al., 2021). A large number of studies point out that N fertilizer can significantly improve the seed yield components. Song et al. (2008) found that N fertilizer could significantly raise the NFTs of E. nutans to improve the number of seeds per plant and thus increase seed yield. Xiao (1984) obtained a similar result, namely that the NFTs    (2008) showed that N fertilizer significantly boosted NFTs and spike weight, thus enhancing seed yield. Investigation of the seed yield of tall fescue accounted for the fact that the amount of N fertilizer application is proportional to NFTs, NFLs and NFFLs per spike within a particular range (Ma et al., 2003). In addition, similar findings have been reported with regard to the seed yield of Lolium perenne, N fertilizer increased NSPs per fertile tiller, NFFLs per spike, and TSW (Hides, Kute & Marshall, 2010;Elgersma, Nijs & Eeuwijk, 1989). In this study, seed yield (r = 0.909, P < 0.05), NFTs/m 2 (r = 0.950, P < 0.05) and TSW (r = 0.921, P < 0.05) were dramatically positively correlated with N fertilizer treatment by means of path analysis ( Fig. 1 and Table 4). This result indicates that N treatment was one of the most effective approaches to improving K. melanthera production by enhancing NFTs and TSW. Furthermore, we also found that seed yield was non-significantly correlated with NSPs per fertile tiller (r = 0.806, P > 0.05) and NFLs per spike (r = 0.818, P > 0.05). These findings are not entirely consistent with other studies. Wang et al. (2005a) researched the influence of five yield components on the seed yield of Dactylis glomerata, and determined the following order of influence: NFTs/m 2 > NFLs/ spike > NFFLs/spike > TSW > NFLs/fertile tiller. However, a different order was obtained for K. melanthera: NFTs/m 2 > TSW > NFFLs/spike > NFLs/spike > NFLs/fertile tiller. The reasons for the discrepancies may be the difference in sampling regions and studied species.

Effect of N on seed quality
The seed quality can be reflected by the standard germination rate (SGR), germination energy (GE), and accelerated aging germination rate (AAGR). The higher the values are for these, the stronger the seed vigor (Lin et al., 2020). It is worth noting that when AAGR is compared to SGR, the aging-accelerated conditions can explain the resistance to adversity that supplies a more reliable reference for seed growth in specific environments such as the QTP (Li & Mao, 2013;Chen et al., 2017). The number of SGR was inferred under suitable environmental conditions and cannot guarantee actual germination rate in the field and survivability under stress, therefore, it is always in synergy with AAGR to evaluate potential viability of seed (Mao, Hou & Wang, 2016). Dehydrogenase (DE) and acid phosphoesterase (APH) activity are often used as crucial indices to test vigor and utilization value of forage seed, because these two indices more accurately reflect the quality and stress resistance of seeds in different stages of growth than seed germination count (Li & Mao, 2013;Chen et al., 2017). Currently, a large number of researchers have shown that N fertilizer is a key factor in increasing seed yield of perennial herb, but the effect of N on pasture seed quality varied with the different species, growing conditions, Figure 3 (continued) presentation seed yield; HSY, harvested seed yield; SGR, standard germination rate; GE, germination energy; AAGR, accelerated aging germination rate; DE, dehydrogenase; APH, acid phosphoesterase; N 1 -N fertilizer 0 kg·hm −2 (Control); N 2 -N fertilizer 60 kg·hm −2 ; N 3 -N fertilizer 90 kg·hm −2 ; N 4 -N fertilizer 120 kg·hm −2 ; N 5 -N fertilizer 150 kg·hm −2 ; N 6 -N fertilizer 180 kg·hm −2 ; N 7 -N fertilizer 210 kg·hm −2 ; N 8 -N fertilizer 240 kg·hm −2 . Full-size  DOI: 10.7717/peerj.14101/ fig-3 harvesting methods, and/or harvesting time (Liu, 2020). The SGR and GE of Medicago sativa (Wang, 2005b) and Festuca elata (Cheng et al., 2003) were significantly increased by N fertilizer application, as well as APH activity of Festuca elata were also significantly upward. The reason may be that N fertilizer can promote the transfer of plant metabolites to reproductive organs and consequently increase plumpness and 1,000-seed weight of plant seed. However, the study of Qiao & Han (2010) demonstrated that N application had no significant effect on seed quality of E. nutans, a native perennial grass in QTP region. The results of this study were in agreement with those results of present study, which the seed quality indices (GR, GE, AAGR, DE and APH activity) of K. melanthera were not significantly affected by N fertilizer in either 2016 or 2017 (Table 2).

CONCLUSION
K. melanthera is a beneficial multi-purpose perennial grass in the eastern QTP region. It is commonly used in the treatment of degraded and sandy grassland, as well as for artificial planting to supply forage for livestock. However, the limited supply and poor quality of seeds have restricted its popularization and utilization in QTP. In this work, a two-year trial in eastern QTP was implemented to evaluate the effects of seed yield and quality of K. melanthera under different N fertilizer levels. The results showed that the optimal N application rate was 180 kg·hm −2 during trial years 2016-2017, and most of seed yield indicators reached the respective highest values at this N level, including the harvested seed yield (HSY), potential seed yield (PSY), presentation seed yield (PRSY), number of tillers (NTs), number of fertile tillers (NFTs), and 1,000-seed weight (TSW). The present findings provide a practical technical groundwork for improving seed productivity of K. melanthera in eastern QTP.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the Sichuan Provincial Key Science and Technology Project (2019YFN0170, 2022YFQ0076). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability
The following information was supplied regarding data availability: